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Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at 2 weeks of ocular hypertension (OHT) relative to non-injured controls (Student’s t-test, n.s.: not significant, N = 5 mice/group). (B) Real-time qPCR analysis confirms that retinal YFP gene expression does not change within 2 weeks of OHT damage (Student’s t-test, n.s.: not significant, N = 5 mice/group). (C-E) RGC co-expressing YFP and SMI-32 are selected for dendritic arbor imaging and 3D reconstruction. Supplementary Figure 2. Analysis of whole-mounted retinas immunolabeled with an antibody recognizing pAMPKThr172 revealed a substantial increase of AMPK activity in RGC axons, visualized with SMI-32, at two weeks after glaucoma induction. Scale bars = 25 μm. Supplementary Figure 3. (A, B) Retinal imaging showed no fluorescein extravasation in eyes injected with compound C or vehicle. (C) In contrast, marked fluorescein extravasation was found in ischemic retinas subjected to central retinal artery ligature. (D), a procedure known to compromise the BRB [31] and used as positive control (Fig. S3C). (D, E) T cell infiltration, visualized with markers the MHCII, MPO and CD45, was not detected in retinas treated with compound C or vehicle. (F) Ischemic retinas, used as positive controls, display cells positive for MHCII, MPO and CD45. (Q) Quantification of cells expressing MHCII, MPO and CD45 confirm lack of cellular infiltrates in compound c-treated retinas. (ANOVA with Tukey’s multiple comparison post-hoc test, * = p

Additional file 1.

Les péricytes sont des cellules contractiles qui s'enroulent le long des parois des capillaires. Dans le cerveau, les péricytes jouent un rôle crucial dans la régulation du diamètre capillaire et du débit sanguin vasculaire en réponse à la demande métabolique. Au cours de l'ischémie, il a été suggéré que les péricytes pourraient resserrer les capillaires, le restant même après la reperfusion et entraîner une insuffisance du reflux microcirculatoire. Malgré cela, on sait peu de choses sur le rôle des péricytes dans les maladies du cerveau et leur contribution à la dynamique capillaire et au débit sanguin local. Afin de mieux comprendre la réponse des capillaires et des péricytes lors d’une lésion ischémique de la rétine et d’identifier les mécanismes moléculaires de la constriction capillaire induite par les péricytes, nous avons caractérisé la réponse des péricytes au cours d’une ischémie rétinienne transitoire ex vivo et in vivo et nous élucidons les mécanismes de contractilité du péricyte ischémie. Nous avons démontré que l'ischémie entraînait des modifications vasculaires anormales telles qu'une réduction générale du diamètre capillaire et une augmentation du nombre de constrictions capillaires à l'emplacement du péricyte, ce qui suggère que l'ischémie favorise une constriction rapide des péricytes sur les capillaires rétiniens, entraînant un dysfonctionnement microvasculaire majeur., Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. It has been suggested that, during brain ischemia, pericytes constrict the capillaries, which remain constricted even after reperfusion resulting in impaired microcirculatory blood flow. Despite this, little is known about the role of pericytes in brain and retinal diseases and their contribution to capillary dynamics and local blood flow. To better understand the response of capillaries and pericytes during ischemic injury in the retina, and to identify the molecular mechanisms of pericyte-mediated capillary constriction, we characterized the response of pericytes during transient retinal ischemia ex vivo and in vivo. We demonstrated that ischemia leads to anomalous microvascular changes characterized by a marked reduction in capillary diameter. This response was accompanied by an increase in the number of capillary constrictions at pericyte locations suggesting that ischemia promotes rapid pericyte constriction on retinal capillaries leading to microvascular dysfunction. Lastly, we show that ischemia increases intracellular calcium in pericytes suggesting that pericyte contraction leading to capillary constriction is a calcium-dependent process.

Increasing evidence indicates that pericytes are vulnerable cells, playing pathophysiological roles in various neurodegenerative processes. Microvascular pericytes contract during cerebral and coronary ischemia and do not relax after re-opening of the occluded artery, causing incomplete reperfusion. However, the cellular mechanisms underlying ischemia-induced pericyte contraction, its delayed emergence, and whether it is pharmacologically reversible are unclear. Here, we investigate i) whether ischemia-induced pericyte contractions are mediated by alpha-smooth muscle actin (alpha-SMA), ii) the sources of calcium rise in ischemic pericytes, and iii) if peri-microvascular glycogen can support pericyte metabolism during ischemia. Thus, we examined pericyte contractility in response to retinal ischemia both in vivo, using adaptive optics scanning light ophthalmoscopy and, ex vivo, using an unbiased stereological approach. We found that microvascular constrictions were associated with increased calcium in pericytes as detected by a genetically encoded calcium indicator (NG2-GCaMP6) or a fluoroprobe (Fluo-4). Knocking down alpha-SMA expression with RNA interference or fixing F-actin with phalloidin or calcium antagonist amlodipine prevented constrictions, suggesting that constrictions resulted from calcium- and alpha-SMA-mediated pericyte contractions. Carbenoxolone or a Cx43-selective peptide blocker also reduced calcium rise, consistent with involvement of gap junction-mediated mechanisms in addition to voltage-gated calcium channels. Pericyte calcium increase and capillary constrictions became significant after 1 h of ischemia and were coincident with depletion of peri-microvascular glycogen, suggesting that glucose derived from glycogen granules could support pericyte metabolism and delay ischemia-induced microvascular dysfunction. Indeed, capillary constrictions emerged earlier when glycogen breakdown was pharmacologically inhibited. Constrictions persisted despite recanalization but were reversible with pericyte-relaxant adenosine administered during recanalization. Our study demonstrates that retinal ischemia, a common cause of blindness, induces alpha-SMA- and calcium-mediated persistent pericyte contraction, which can be delayed by glucose driven from peri-microvascular glycogen. These findings clarify the contractile nature of capillary pericytes and identify a novel metabolic collaboration between peri-microvascular end-feet and pericytes.

Table S1. Summary of experiments and comparisons. The table summarizes all experiments performed including the treatment groups and number of mice used. Where appropriate, analyses, statistical comparisons, mean values (±SEM), and P values are also indicated. Please note that some animals/retinas were used for more than one experiment, therefore, the total number of mice is less than total number of experiments. (DOCX 45 kb)